Role of a distal promoter element in the S‐phase control of the human H1.2 histone gene transcription

Abstract

The expression of one of the human main type H1 histone genes (termed H1.2) appears to be regulated by several trans-acting factors. Upstream of consensus regulatory regions, such as the TATA-, CCAAT-and H1-box (AAACACA) sequences, a crucial control site is located between nucleotide positions -536 and -412 (relative to the ATG initiation site). Removal of this promoter portion causes in chloramphenicol acetyl transferase reporter gene constructs a loss of the S-phase control function of the H1.2 promoter in HeLa cells. Electrophoretic mobility-shift assay and DNase I footprinting analysis suggest that the H1-box variant AAACAGA is a potential control element within the distal promoter region.The H1 group of mammalian histones comprises several subtypes, which differ in primary structure. Developmental control and cell-type-specific composition of the H1 complement has been described [l]. Compared with core histones, H1 proteins represent the most variable class of the otherwise highly conserved histone protein family. Five main type H1 histone genes have been described [2-51 in addition to genes which are solely expressed in specific cell types, such as the H1° gene [6], which is only expressed in highly differentiated cells [7, 81, and the Hlt gene [9, 101, which is transcribed during the spermatocyte stages of spermatogenesis [111.Despite the different transcriptional regulation, all vertebrate H1 genes (except the avian red blood cell H1 subtype H5) share a promoter element with the sequence AAACACA [12, 131. Its involvement in the S-phase-dependent transcription of chicken [12] and human [13] H1 genes has been demonstrated by deletion analysis [12] and factor titration throughout the cell cycle [13]. We have extended the functional analysis of the human H1.2 (for nomenclature, see [2]) gene promoter including more distal promoter sites. A combination of a series of promoter deletions, reporter gene constructs and footprint analysis revealed that promoter sites upstream of the consensus H1 box contribute to the S-phasespecific expression of the H1.2 gene. MATERIALS AND METHODS Cell cultureHeLa cells were grown in minimal essential medium at 37°C with 10% (by vol.) fetal calf serum in the presence of 5% CO,. The cells were grown to a density of 3-4X105 cells/ml before harvesting. Subsequently they were rinsed with NaCW, (0.14 M NaC1, 2.5 mM KC1, 8.1 mM Na2HP04, 1.5 mM KH2P04, pH 7.4) and treated with trypsinBDTA [0.05% trypsin (mass/vol.), 0.02% EDTA (mass/vol.), in Puck salt (solution A)] for 2-3 min at 37°C. 16-20 h prior to transfection, cells were seeded at a density of 4-6X104 cells/cm2 in order to reach logarithmic growth at the time of transfection. Cloning and stepwise deletion of 5' flanking sequences of the human H1.2 histone geneThree fragments (termed KO, K1 and K2, see Fig. 2) were excised by restriction enzyme cleavage from the H1.2 promoter [4] and inserted into a Bluescript KS vector, which had been cut with EcoRI and HincII. After insertion into Bluescript vector, XbaVXhoI-flanked fragments ...

HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

“…In proliferating mammalian cells, expression of histones peaks in S phase of the cell cycle as a result of both increased gene transcription and posttranscriptional regulation (4,9,20,53,58). Cis-acting promoterspecific sequences (or subtype-specific consensus sequences [SSCEs]) in the gene promoters mediate cell cycle periodicity of transcription (3,9,15,27,36). For example, in the promoter of at least one copy of the human histone H2B gene an Oct-1 site is required for S-phase-specific activation, although Oct-1 DNA binding activity remains unchanged from G 1 to S phase (27,51).…”

Conserved sequence elements in human main type‐H1 histone gene promoters : their role in H1 gene expression

“…These reports described an upstream conserved sequence (UCE I [28]) that contained the CH1UE motif. Furthermore, the promoter region described by Eilers et al [35] as a control site in the human H1.2 gene promoter contains the TGTGTTA motif. Further comparison of several mammalian and non-mammalian H1 gene promoters revealed that this sequence element is highly conserved in most higher eukaryotes.…”

“…5, the mutation of just the CH1UE resulted in a reduction by about 50% of the S-phase response mediated by the H1.3 promoter. As a control for this experimental design, a histone full-length H1.2 promoter construct (1.9 kb of the 5′-flanking region ligated to the luciferase reporter gene) previously described to be involved in S-phase control of transcription [35] was analyzed in parallel. It showed about the same ratio as the H1.3 full-length construct between the reporter gene activity in S-phase cells compared with cells remaining in the blocked state.…”

“…The H1.3 promoter (as for the other main type-H1 gene promoters except H1.1) features both general and H1-specific consensus regulatory elements. At nucleotide position Ϫ27 (relative to the start site of transcription), a TATA box is located, which is separated by 17 bp from a CCAAT box at position [52]; H1.1 [14]; H1.2 [35]; H1.3 [11]; H1.4 [11]; H1.5 [12]; and H1t [15] Ϫ49. The exact distance of 17 bp between these two consensus elements is a general feature of the main-type human H1 promoters.…”

“…In addition, the CH1UE motif was mutated within the Ϫ587 construct and the luciferase activity was determined in S-phase and non-S-phase cells. The H1.2 promoter [35] was included in the assay as a control. Cells were first transfected with the respective reporter gene constructs and were then blocked at G1/S transition by aphidicolin.…”